Bioremediation - Rhizoremediation /Rhizodegradation

1. RHIZOREMEDIATION
DEPARTMENT OF MICROBIOLOGY
M.KEJAPRIYA,
I – M.Sc., MICROBIOLOGY,
VIVEKANANDHA ARTS AND
SCIENCE COLLEGE FOR
WOMEN,
VEERACHIPALAYAM, SANKAGIRI.
SUBJECT: BIOREMEDIATION
GUIDED BY:
Dr.R.DINESH KUMAR,
ASSISTANT PROFESSOR,
DEPARTMENT OF MICROBIOLOGY
VIAAS, SANKAGIRI.

2.  The ever increasing pollution levels have become a major
environmental concern. The rapid technological
advancements, industrialization, massive use of fertilizers and
pesticides etc. have added on to the baggage of pollutants.
 These pollutants include: hydrocarbons, heavy metals, dioxins,
chlorinated compounds etc.
 Various conventional physico-chemical techniques like
composting, land forming have been used to combat this
problem. These are all invasive, time consuming, lead to
generation of more toxic substances and even result in
emission of greenhouse gases. Therefore, biological
remediation of these pollutants is a viable option.

3.  Bioremediation involves the use of living organisms to
neutralize these pollutants or to get rid of them.
 One such bio remedial technique which has come into light is
rhizoremediation. Over the past few years intensive studies are
being carried to investigate various microorganisms as bio
remedial agents.
 Rhizoremediation is one such technique involving the use of
rhizobial microflora mainly rhizobacteria. It is a technique
trending these days and is ecofriendly and even cost effective
and offers impactful future prospects.

4.  Rhizosphere is nutrient rich area or zone surrounding the plant
roots. It is rich in microorganisms mainly characterized as plant
growth promoting organisms which enhance plant growth directly or
indirectly.
 These rhizobial microbes show immense interactions with the plants
and are also involved in the remediating of the harmful pollutants
found in the soil as poly-aromatic hydrocarbons, polychlorinated
biphenyls, halogenated compunds, pesticides like atrazine, heavy
metals like cadmium, mercury and many more.
 This clean-up of the hazardous wastes and chemicals brought about
by rhizomicroflora is known as rhizoremediation.

5.  Rhizoremediation is a specific subset of phytoremediation. Its cost
effectiveness, convenience with little or no side effects have made it
a growing technology. It is a clean and green phenomenon.
 Although, it can be time consuming, yet its end results are
phenomenal as the pollutant is completely destroyed or it is
converted to harmless form. It is suitable for large or small sites with
low to moderate pollutant levels.
 It is a symbiotic relationship between the plant roots and the
microorganisms where plant roots provide rich niche for the growth
of the microbes and in turn microbes as the biocatalyst that remove
the pollutants,
 Even the utilization of non-rhizopsheric or ex-situ microbes can help
in remediatng the polluted sites but these organisms face certain
shortcomings compared to rhizospheric microflora.

6.  The shortcomings include lack of nutrients in soil, soil surface
properties, toxicity or reduced bioavailability of the contaminants,
competition with the indigenous microflora and failure to express
required catabolic functions etc.
 Moreover, the number of microbes in the rhizosphere out number the
microbes in bulk soil. Increased degradation of the pollutants in the
rhizosphere is beneficial to the plant and result in improved plant
growth on contaminated soil.

7.  Plant have a basal role in the process: As plant roots can be
considered as a substitute for tilling the soil, to spread the root
associated microorganisms through the soil and to penetrate layers
normally not accessible to bacteria or other microbes and to
incorporate nutrients, bring oxygen and provide better redox
conditions which help to stimulate and activate the rhizosphere
microflora. Also, majority of the plants live in symbiotic relationship
with mycorrhiza which act as web like extension of the root system
enhancing the absorptive area of plants.
 Plants also offer an effective clean-up technology compared to
conventional methods. They provide their own solar energy by
photosynthesis and pump up pollutants with water stream making it
much cheaper.
 It is in-situ technology. It generates less or even no waste and instead
creates economic benefits.

8. PLANT AND
MICROBES
INTERACTIONS
IN THE
RHIZOSPHERE
ROOT
COLONIZATION
MAINTENANCE
AND REGULATION
OF GENE
EXPRESSION
COMMUNICATION
AND DYNAMICS

9.  The bio-degradative microorganisms colonize the rhizosphere
similar to other rhizospheric microbial populations. These can either
be recruited from the soil reservoir during growth of the plants on
contaminated soil or these originate from (bacterized) seeds.
 Microorganisms are attracted towards the root exudates (secretion of
plant roots) by chemotactic movement and they subsequently spread
through the soil during root emergence and growth.
 Using in-vivo expression technology (IVET), transcriptomics and
mutants defective in motility, the mechanism of root colonization
and identification of genes which are activated during rhizosphere
colonization are being discovered.
 Successful rhizosphere colonization depends not only on the
interactions between the plants and the microbes but also on the
interactions with other rhizospheric microorganisms and the
environment.

10.  Selection of the appropriate plant to stimulate contaminant
degradation is a key aspect in the rhizoremediation strategy, however
choice of the bio-degradative microflora is of major concern.
 Plant roots release compounds called as root-exudates as sugars,
organic acids, fatty acids, secondary metabolites, nucleotides and
also inorganic compounds that play an important role in establishing
and determining the rhizosphere microbial population.
 These help in selection of the microbes which respond to the exudate
buffet rapidly. These secretions help in determination and regulation
of the concerned bio-degradative microorganisms either directly or
indirectly.

11.  Plants and the microorganisms send and receive multiple signals
responsible for recognition of microbes, recruitment of catalytic
potential, mycorrhization, resistance to stresses and quorum sensing.
 Various biotic factors and abiotic factors including the growth stage
of the plant, soil type, contaminant type, season, temperature and the
density of other microorganisms etc. play a significant role in
shaping this interaction.
 Example: Siciliano et al., demonstrated that catabolic alkane
monooxygenase gene were more prevalent in rhizospheric microbial
communities than in the bulk soil contaminated with hydrocarbons.

12. FACTORS AFFECTING
RHIZOREMEDIATION
Soil
conditions
Temperature
pH
Soil organic
matter
Microbial
diversity
Plants
Bioavailability
of pollutants
present in soil

13.  Rhizoremediation is affected by various physical, chemical and
biological factors. These may include temperature, pH, soil
conditions, microbial diversity, aeration, organic matter content, rate
of exudation, age of the plant, availability of nutrients and the
presence of contaminants.
 These factors control the effectiveness of the process in one way or
the other as follows:
SOIL CONDITIONS:
The accomplishment of rhizoremediation depends on the soil
physico-chemical conditions properties such as moisture, redox
conditions, temperature, pH, organic matter, nutrients and nature and
the amount of clay etc. that affect the microbial activity as well as
diffusion of the chemicals in the soil.

14. TEMPERATURE:
Temperature and pH are the principle factors affecting the bio-degradation of
contaminants of various types in the soil. It not only has an impact on the rate of
biochemical reactions but also has an impact on the microbial activity.
It is essential that contaminated sites be at the optimum temperature for
bioremediation to progress successfully, since excessively high or low temperatures
sometimes inhibit microbial metabolism. In addition, the solubility and bioavailability
of a contaminant will increase as temperature increases, and oxygen solubility will be
reduced, which will leave less oxygen available for microbial metabolism.
Until recently, most research has focused upon the biodegradation of organic
compounds at mesophilic temperatures; however, evidence suggests that the indigenous
microbial communities of a contaminated site will adapt to the in situ temperature, such
as those in Arctic and Antarctic soils.
For examples, a selection of Rhodococcus species that were isolated from an
Antarctic soil were successfully degrade a number of alkanes at -2°C but were severely
inhibited at a higher temperature.

15. In addition, the PAHs naphthalene and phenanthrene were successfully degraded
from crude oil in seawater at temperatures as low as 0°C.
pH:
The biodegradation of the contaminant is dependent on specific enzymes secreted
by the microorganisms. As the enzymes are pH dependent therefor, all microbes
require an optimum pH for their growth viz., bacteria grow at the optimum pH values
of 6.5 – 7.5.
Singh et al., (2006) found that biodegradation of the organophosphate pesticides
was slower in lower pH soils compares to neutral and alkaline soils.
SOIL ORGANIC MATTER:
It has a considerable effect on the degradation of the contaminants in soil as the
soil organic matter acts as rich source of nutrients for growth of bio-degradative
microflora and it also controls the movement of contaminants by absorption and
desorption processes.
Perrin-Ganier observed that biodegradation of isoproturon (herbicide) was
enhanced by adding sewage sludge (source of organic matter).

16. MICROBIAL DIVERSITY:
Rhizospheric microbial populations enhance the process of rhizoremediation by
increasing the humification of organic pollutants. eg: a microbial consortium degrades
the pollutants much more effectively than a single strain/species because of the
presence of partners, which use various intermediates of degradation pathway more
efficiently(Kuiper et al.,). Many such microbes colonizing the plant roots have been
identifies as Pseudomonas spp., Bacillus spp., Azotobacter and many more.
PLANTS:
The age of plant influences the nature of root exudates which in turn characterizes
the microbial flora residing in rhizosphere and bringing about the process of
rhizoremediation. Also, plant roots have major role and help in determination of
microbial diversity around them.
BIOAVAILABILITY OF POLLUTANTS PRESENT IN SOIL:
The nature of pollutants present in the soil also governs the microbial populations
found in it, thereby affecting the rate of process. Bioavailability is also extremely
important for the biodegradation of organic pollutants.

17. It is frequently observed that the rate of removal of compounds from soils is very
low even though the coumpounds are biodegradable. However, the substrates in these
instances may not be in a form that is readily available to the microorganisms.
Biodegradation of hydrophobic pollutants may take place only in the aqueous
phase; for example, naphthalene is utilized by pure cultures of bacteria only in the
dissolved state.
Bouchez et al.,(1995) similarly showed that phenanthrene biodegradaion occurs
only in the aqueous phase. The many observations of linear growth of bacteria and
yeasts on slightly soluble substrates may be explained by the need for the substrates to
be in the aqueous phase.
In the case of the PAHs, it has been shown that linear growth is due to mass transfer
limitation from the solid phase to the liquid phase.

18. BIO-
SURFACTANT
PRODUCTION
BIOFILM
FORMATION
ORGANIC
ACID
PRODUCTION

19.  The microorganisms carry out the process of biodegradation by
various mechanisms:
BIO-SURFACTANT PRODUCTION:
Bio-surfactants are amphiphilic molecules (both hydrophobic and hydrophilic)
which form micelles with the hydrophobic compounds as poly-aromatic hydrocarbons
and thereby increase their bioavailability for the purpose of bioremediation.
The micelles are formed when the surfactant concentration exceeds a certain
critical value i.e specific for the compound. The micelle formation enhances the
solubility of hydrophobic contaminants and thus their degradation by microbes.
Kuiper et al., (2004) isolated Pseudomonas putida from plant roots at a site
polluted with PAHs that produced two lipopeptide bio surfactants.
BIOFILM FORMATION:
Biofilm is the aggregate of microorganisms in which cells are freqently embedded
within a self-produced matrix (of exopolysaccharide).

20. The microflora in biofilm have better chances of survival as they are protected by
matrix and thus help in immobilization and degradation of the contaminants. (Segura et
al., (2009)
ORGANIC ACID PRODUCTION:
Organic acids as phytic acid, gluconic acid etc. secreted by various rhizobial
microflora lower the pH of soil, thereby increasing the solubility of contaminants and
enhancing their degradation.
Various other mechanisms are involved in rhizoremedial action of microorganisms
and may include siderophore production, release of enzymes like oxidoreductases,
ACC deaminase production and many more (Wu et al., 2006).

21.  Presence of bio-degradative strains, the expression of the catalytic
genes and maintenance are all crucial factors which determine the
success of rhizoremediation. However, under natural conditions, the
phenomenon can be slow, indicating that some of these factors or
others are limiting the removal of pollutants (Thijis and
Vangronsveld 2014). Therefore, technologies for improving the
efficiency of the process were developed which include:
BIO-STIMULATION:
It is the modification of the environment to stimulate the existing microorganisms
for carrying out the rhizoremedial mechanism. It involves addition of compost as
fertilizer, addition of minerals like phosphorus and nitrogen as bait for the microbes,
improving physico-chemical properties of the soil by using additives as bio-surfactants
for oil degradation etc.

22. BIO-AUGMENTATION:
It is defined as the introduction of microbial strain or consortium into the soil to
enhance degradation process. Delivery methods may involve seed coating, soil
drenching, root dipping etc.
GENETICALLY ENGINEERED MICROORGANISMS:
For certain pollutants no catabolic pathways exist among the microbes to promote
their degradation. To counter such pollutants genetically engineered microbes are
being used.
Barea et al., (2005) reported the improved remediation of toluene by using
genetically engineered bacteria. They transferred pTOM plasmid which encodes
toluene degradation genes via conjugation from B.cepacia G4 to B.cepacia L S 2 4.
RHIZOENGINEERING:
It involves the use of transgenic plants which secrete more root exudates which all
indirectly have a significant effect on rhizosphere microflora. All these techniques help
to improve rhizoremediation thereby making it more promising future technology.

23.  Rhizoremediation is great for the removal or harmful organic
compounds and pollutants like petroleum, hydrocarbons etc.
 They completely degrade these compounds to very small particles or
minerals that are then easily removed from the soil.
 It is an eco-friendly and sustainable method of treating contaminated
soil and water as no other extra substance is used to treat the soil
thereby preserving its quality and improving its fertility and health at
the same time.
 It is an integrated way of dealing with soil contamination as only
plants are used along with the natural existing microorganisms in the
root zone of the plant (rhizosphere).
 This is a long term benefit since as long as the rhizosphere is active,
there will be continuous recycling of nutrients and removal of toxic
substances from the soil at no external cost.

24.  Some more persistent pollutants like substances with high molecular
weight (substance made of many polymers) and high number of
chlorine attached to it cannot be degraded or removed from the soil.
Thus, this process only works for mild to moderately contaminated
soil. Severely contaminated soils cannot be treated via
rhizoremediation.
 Sometimes, during rhizoremediation, instead of removing the toxic
substance from the soil, it is possible that a more toxic and harmful
substance is created which cannot be removed from the soil. For this
reason, it is essential to which plants can work to remove what kind
of pollutants from the soil and vice versa.